Leishmania parasites are opportunistic protozoan flagellates that are the causative agents of devastating and often fatal diseases in much of the tropical and subtropical world. An increasing problem is the occurrence of Leishmania/HIV co-infection in immunocompromised individuals where persistent and previously asymptomatic parasites develop leishmaniasis after the outbreak of AIDS. In these protozoan flagellates, myo-inositol plays an especially important role as the precursor for GPI-anchored protective and/or immunomodulatory surface molecules, which are several orders of magnitude more abundant on the surface of these parasites than in the mammalian host. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway. For inositol salvage, Leishmania donovani has an active myo-inositol/H+ transporter (MIT) that is driven by a proton-electrochemical gradient across the parasite membrane. Moreover, the proton-coupled Leishmania MIT is functionally and structurally unrelated to the human sodium-coupled myo-inositol transporters (SMIT1 and 2) in the intestine and kidney. Many of the transporters in Leishmania and other protozoan parasites are thought to function as proton-coupled active transporters, but these carriers are in general not well characterized at the molecular level. Leishmania MIT has an exceptionally high substrate specificity, in contrast to the human inositol transporters, and the C-2, C-3, and C-5 hydroxyl groups of myoinositol are critical for substrate recognition by the Leishmania permease. Three specific aims will investigate the structure-function relationship of the L donovani MIT as a promising target for delivery of cytotoxic inositol analogues, and as a model active transporter in these early protozoan eukaryotes. (i) In the first specific aim chimeras between MIT and the structurally related E. coli xylose/H+ symporter (which does not transport myo-inositol) will be generated to investigate the domain(s) in MIT that are responsible for substrate selectivity. (ii) In the second specific aim part of the substrate permeation pathway of MIT will be mapped by cysteine scanning mutagenesis of transmembrane domain 1 (TM1) that contains the functionally essential residues Asp19. These experiments will test the hypothesis that TM1 forms part of the substrate permeation pore that allows active and selective myo-inositol transport across the parasite membrane. Hence the first two specific aims will probe the "active site" of this permease that allows inositol recognition and subsequent transport across the plasma membrane, (iii) MIT belongs to a large sugar transporter superfamily of membrane transporters with 12 transmembrane domains that are thought to have evolved through gene duplication of an ancestral 6-transmembranedomain transporter. Hence the final specific aim will test the hypothesis that either the MIT N-terminal or C-terminal 6 transmembrane segments (N6 or C6, respectively) are sufficient for transport function, similar to an ancestral 6- transmembrane-domain transporter. Functional characterization of the MIT half-transporters N6 or C6 will be performed in the Xenopus oocyte expression system and in an MIT-less L. donovani strain that was recently developed in the laboratory, following transfection with each MIT half-transporter individually, or with N6-N6 and C6-C6 tandem repeat chimera constructs.